Evidence for mitochondrial localization of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea in human colon adenocarcinoma cells.
N-(4-Methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) is a new agent that exhibits high therapeutic activity against human and rodent tumor models. Initial studies indicated that in vitro [3H]MPCU was concentrated 4- to 6-fold in GC3/c1 human colon adenocarcinoma cells in an azide-sensitive manner. In this study the dependence of uptake and concentrative accumulation of MPCU upon temperature, plasma membrane potential, and the electrochemical potential of mitochondria has been examined. Accumulation and efflux of MPCU were temperature dependent. At 3.6 microM MPCU, initial rates of uptake (15 s) were 1.4, 38.0, and 84.2 pmol/min/10(6) cells at 2 degrees C, 23 degrees C, and 37 degrees C, respectively. The rate of uptake and concentrative accumulation within GC3/c1 cells was not altered in high K+ buffer or by 1 mM ouabain, indicating that plasma membrane potential was not significant in these processes. Concentrative accumulation, but not initial uptake, was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol, and sodium azide. Glucose partially antagonized the inhibition of these agents which uncouple oxidative phosphorylation. Oligomycin, an inhibitor of mitochondrial ATP synthase, did not inhibit uptake or concentrative accumulation of MPCU. However, oligomycin in the presence of 2-deoxyglucose significantly inhibited concentrative accumulation of MPCU. These results suggested that concentrative accumulation of MPCU was dependent upon the mitochondrial transmembrane gradient rather than ATP, although direct implication of ATP could not be excluded. To examine which component of this gradient was predominant in causing MPCU sequestration, the ionophores valinomycin and nigericin were used. Valinomycin, which collapses the charge gradient across the mitochondrial matrix membrane, caused only slight inhibition of MPCU accumulation, and the effect was similar at 2 or 10 mumol. In contrast, nigericin (which collapses the pH gradient and increases mitochondrial membrane potential) inhibited by approximately 90% concentrative accumulation of MPCU. These data suggested that MPCU was being concentrated in mitochondria and that this was dependent upon the pH gradient across mitochondrial membrane. In cells exposed to MPCU or the analogue N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, enlargement of mitochondria was observed within 24 h and appeared to be the initial morphological change associated with drug treatment. These results implicate mitochondria as a site of sequestration of diarylsulfonylureas and as a potential site of action.
Houghton
P J
PJ
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318.
Bailey
F C
FC
Houghton
J A
JA
Murti
K G
KG
Howbert
J J
JJ
Grindey
G B
GB
D000230:Adenocarcinoma / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation; D003110:Colonic Neoplasms / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D009550:Nigericin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D013453:Sulfonylurea Compounds / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
D000970:Antineoplastic Agents; D009840:Oligomycins; D013453:Sulfonylurea Compounds; D014475:Uncoupling Agents; D014634:Valinomycin; C062821:LY 181984; D010042:Ouabain; D009550:Nigericin